Annotation of the results obtained in 2023
The terminal respiratory quinol oxidase CydABX of E. coli couples the oxidation of ubiquinol or menaquinol with molecular oxygen to the formation of a proton motive force. This membrane energy currency is then used by the bacterial cell to synthesize ATP through the mechanism of oxidative phosphorylation. In addition to functioning as a molecular energy transducer, CydABX is involved in other vital processes in the E. coli cell. In particular, this quinol oxidase contributes to the mechanisms of maintaining optimal redox balance in the bacterial cell by neutralizing reactive oxygen species such as hydrogen peroxide. Since CydABX belongs to the superfamily of terminal oxidases that are found only in prokaryotic organisms, including pathogenic ones, these enzymes may serve as promising therapeutic targets. In the course of the work on the second stage of the project, new important scientific data on the redox transformations of the studied enzyme were obtained. Carrying out studies using the fast mixing method using a diode array spectrophotometer made it possible to record the absorption spectra of CydABX with millisecond time resolution. A comparative analysis of the kinetics of redox reactions of the mutant form of the oxidase and the wild-type enzyme has made it possible to significantly advance our understanding of the molecular mechanism of CydABX functioning, in particular, the mechanism of intraprotein electron transfer. Based on the results of the project obtained during its implementation, in 2023 three papers were published in the journals indexed in the Web of Science Core Collection and Scopus databases: 1. Azarkina N.V., Borisov V.B., Oleynikov I.P., Sudakov R.V., Vygodina T.V. (2023) Interaction of terminal oxidases with amphipathic molecules. International Journal of Molecular Sciences, 24, 6428. https://doi.org/10.3390/ijms24076428 2. Borisov V.B. (2023) Generation of membrane potential by cytochrome bd. Biochemistry-Moscow, 88, 1504-1512. https://doi.org/10.1134/S0006297923100073 3. Borisov V.B., Nastasi M.R., Forte E. (2023) Cytochrome bd as antioxidant redox enzyme. Molecular Biology, 57, 1077-1084. https://doi.org/10.1134/S0026893323060031

 

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Annotation of the results obtained in 2022
Deciphering the mechanisms of action of energy-converting terminal quinol oxidases of bacteria at the molecular level is an important fundamental problem of modern biochemistry and biophysics. The solution of this problem will contribute to the search for new pharmaceutical drugs against a number of microbial infections, given the significant contribution of the quinol oxidases to the resistance mechanisms of pathogenic microorganisms and the absence of these enzymes in humans. In the course of the work on the first stage of the project, important scientific results on real-time redox transformations of the E. coli CydABX quinol oxidase were obtained. The catalase-like activity of the E. coli AppBCX quinol oxidase was also studied and its inhibitory analysis was carried out. Preparations of the isolated solubilized AppBCX have been found to scavenge H2O2 at a high rate, producing O2. The addition of H2O2 to the same buffer that does not contain the enzyme or contains thermally denatured AppBCX does not lead to the production of O2. The latter observation excludes the participation in the reaction of extraneous transition metals associated with the protein. The H2O2-induced O2 production is not inhibited by N-ethylmaleimide (a compound that binds sulfhydryl groups), antimycin A (a compound that specifically binds to the quinol binding site), and CO (a diatomic gas that specifically binds to the reduced heme d). However, the formation of O2 is effectively inhibited by cyanide. Azide is also able to suppress the catalase-like reaction. The addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate AppBCX and does not affect its O2 reductase activity. Based on the obtained data, the mechanism used by AppBCX to carry out the catalase-like reaction was proposed. It was concluded that the ability of the E. coli AppBCX to neutralize H2O2 may play a role in the physiology of bacteria, making them resistant to peroxide-mediated stress. Finally, the important scientific results pointing to a new physiological mechanism for regulating the activity of the mitochondrial cytochrome c oxidase have been obtained. Based on the results of the project obtained during its implementation, in 2022 four papers were published in the journals indexed in the Web of Science Core Collection and Scopus databases: 1. Forte E., Nastasi M.R., Borisov V.B. (2022) Preparations of terminal oxidase cytochrome bd-II isolated from Escherichia coli reveal significant hydrogen peroxide scavenging activity. Biochemistry-Moscow, 87, 720-730. https://doi.org/10.1134/S0006297922080041 2. Borisov V.B., Forte E. (2022) Bioenergetics and reactive nitrogen species in bacteria. Int. J. Mol. Sci., 23, 7321. https://doi.org/10.3390/ijms23137321 3. Friedrich T., Wohlwend D., Borisov V.B. (2022) Recent advances in structural studies of cytochrome bd and its potential application as a drug target. Int. J. Mol. Sci., 23, 3166. https://doi.org/10.3390/ijms23063166 4. Oleynikov I.P., Sudakov R.V., Azarkina N.V., Vygodina T.V. (2022) Direct interaction of mitochondrial cytochrome c oxidase with thyroid hormones: evidence for two binding sites. Cells, 11, 908. https://doi.org/10.3390/cells11050908

 

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